Our long-term goal is to understand how potassium (K+) channels operate in the heart in health and disease. This application for renewal focuses on regulation of K2P and Kv4 channels, key contributors to cardiac IKp and Ito currents. The proposal gains direction from the last period when protein, lipid and drug regulators of these channels were identified and relationships between cardiac K+ channel dysfunction and arrhythmia were further elucidated. Four specific aims consider: what controls surface expression of the channels (Aim 1); how they are modulated once at the surface (Aim 2); isolation of inhibitors and activators to delineate roles of the channels in native cells (Aim 3); and, the structural basis for regulated function as revealed by combined utilization of electrophysiology, biochemistry and electron microscopy (Aim 4). The proposal exploits classical and novel strategies made more powerful by recent advances in biotechnology. In the last period, studies of channels formed by subunits with 2 P domains moved from identification and cloning to in-depth characterization. Formal names ("K2P channels and KCNK genes") were granted and salient attributes made clear: these are K+ selective "leaks" (active across the physiological voltage range) that open and close (gate) and are strictly-regulated. Control is exerted through regulation of channel number and location (N), open probability (Po) and, unitary current (i). Thus, cytosolic proteins control surface expression; phosphorylation and free fatty acids regulate single-channel gating and rectification; and, external proton, toxins and tissue factors modulate flux. Also revealed were roles for the channels in cardiac (and CNS) function and as targets for anti-ischemic drugs and volatile anesthetics. Similar regulatory events determine function of Kv4 voltage-gated channels we find amenable to structural study. Our motivation is that K2P and Kv4 channels operate in strictly-regulated fashion to influence cardiac activity in health while their altered function can lead to disease. These outcomes result because cardiac function requires K+ channels at the correct locale, abundance, and activity level to meet demand. We seek to study these regulatory events in mechanistic and structural detail. Supporting feasibility and significance are findings in the last period and exciting preliminary data.